Cross-presentation of exogenous antigens by host professional antigen-presenting cells (APCs) plays a pivotal role in the initiation and development of T-cell immune responses to tumor associated antigens, including self or mutated self-antigens derived from tumor cells, and foreign antigens derived from infectious agents. Prospective cancer vaccines have been developed that attempt to harness the cross-presentation of exogenous antigens to illicit a specific immune response against a tumor.
However, specific active immunization with cancer vaccines has not been very effective in animal models or in clinical trials (Rosenberg et al., Nat. Med. 10:909-15, 2004.). The primary obstacle to the success of cancer immunotherapy has been the inability of the vaccine to induce a large initial expansion and then persistence of tumor-reactive CTL (Rosenberg et al., Nat. Med. 10:909-15, 2004). Another obstacle to currently available methods of cancer immunotherapy is that potential tumor rejection antigens are not known for most cancers, with the exception of melanoma, and the dominant tumor rejection antigens are likely tumor or patient-specific.
Previously, the inventors have determined that reducing or inhibiting cellular protein degradation with a proteasome inhibitor may result in cellular accumulation and secretion of defective ribosomal products (DRiPs) and short lived proteins (SLiPs) (as well as immunogenic fragments thereof) into “blebs”. It is shown herein that DRibbles released from, or contained within, cells (such as tumor or pathogen infected cells) after proteasome inhibitor-induced autophagy may accumulate DRiPs and SLiPs (and fragments thereof) in autophagy bodies and induce a strong immunity (such as anti-tumor or anti-pathogen) via cross-priming. For example, tumor-derived DRibbles, as well as Antigen Presenting Cells (APCs) (such as dendritic cells) loaded with tumor-derived DRibbles, may activate tumor-reactive Cytotoxic T-Lymphocytes (CTL) and Helper T-Lymphocytes (HTL) more efficiently than APCs incubated with live or killed tumor cells, both in vitro and in vivo. In particular examples, administration of DRibbles to a subject (for example as an isolated DRibble population, as a population of cells producing DRibbles, or as DRibble-loaded dendritic cells) increases the generation of CD4 and CD8 cells and promotes the production of inflammatory cytokines or chemokines, such as one or more of IL-6, IL-12, and TNF-α.
The inventors have previously disclosed methods for producing and isolating Dribbles (US 2009/0220530). The method includes contacting the target cell with a sufficient amount of proteasome inhibitor under conditions sufficient for producing DRibbles, such as conditions that substantially inhibit protein degradation in the cell. In some examples, the method further includes contacting the target cell with a sufficient amount of an autophagy inducer before, during or after contacting with the proteasome inhibitor. The cell may also be contacted with one or more agents that decrease glycosylation of proteins. In one example, the cell is contacted with sufficient amounts of a proteasome inhibitor (such as 20 nM Velcade), an autophagy inducer (such as rapamycin or HBSS) and NH4Cl under conditions sufficient to stimulate production of Dribbles by the cell. In one example, the DRibbles produced under said conditions are harvested by separating said DRibbles from the cell. In one exemplary method, a cell may be contacted with a sufficient amount of a composition that may include a proteasome inhibitor under conditions sufficient to substantially inhibit protein degradation in a cell, for example an incubation of 6-24 hours. The cells may then be incubated under conditions sufficient to induce autophagy in the cell, for example an incubation of 6-24 hours with an autophagy inducer. The resulting cells and DRibbles may then be centrifuged under conditions such that the cells are pelleted while the DRibbles remain in solution. The supernatant containing the DRibbles may then be removed and may then be centrifuged under conditions sufficient to pellet the DRibbles.
However, the inventors herein have recognized that the methods for production and isolation of DRibbles disclosed in the prior art may be insufficient to produce and isolate an enriched population of autophagosomes and their component material to be further utilized as an effective vaccine. The present disclosure describes a novel method for screening cell lines (including both cancer and non-cancer cell lines) that may produce effective vaccines. The cell lines may be characterized as producing autophagosomes that may contain Toll-like receptor (TLR) ligands, damage associated molecular patterns (DAMPs), and molecular chaperones in addition to DRiPs, SLiPs and other tumor antigens when treated with proteasome inhibitors and NH4Cl. Additionally, the cross-presentation of tumor antigens contained within DRibbles may be insufficient to produce an effective immune response against a tumor when presented to a general population of APCs. The present disclosure describes methods for effective cross-presentation of antigen contained within enriched autophagosomes by presenting the enriched autophagosome composition to a specific subset of Dendritic Cells (DCs) that express CLEC9A to produce an efficient immune response.
The inventors herein have recognized the above problems and developed compositions and methods to at least partially address the problems. In one example, a composition, comprising: an enriched population of autophagosomes derived from a non-small cell lung carcinoma cell line, and wherein the enriched population of autophagosomes includes: one or more toll-like receptor agonists; one or more tumor antigens; and one or more damage-associated molecular pattern molecules. In this way, an off-the-shelf vaccine may be available to be administered in order to stimulate a targeted immune response in patients bearing different tumor types.
In another example, a method of inducing a specific immune response in a mammal, comprising: providing a composition comprising: an enriched population of autophagosomes derived from a cell line, the enriched population of autophagosomes including: one or more toll-like receptor agonists; one or more tumor antigens; and one or more damage-associated molecular pattern molecules. In this way, an off-the-shelf vaccine may be administered to patients bearing different tumor types, while supplemented with an inflammation inducing adjuvant derived from the patient's own blood cells.
In yet another example, a method for screening cells that produce allogeneic autophagosome enriched compositions able to induce expression of a selective marker on a subpopulation of peripheral blood mononuclear cells, comprising: contacting a cell with a proteasome inhibitor; contacting the cell with a lysosome inhibitor; harvesting the resulting autophagosomes; determining a molecular signature of the resulting autophagosomes; and selecting cells that divert one or more Toll-like receptor agonist and/or one or more molecular chaperones to the autophagosomes. In this way, toll-like receptor agonists, tumor antigens, and damage-associated molecular pattern molecules may be packaged in an allogeneic autophagosome enriched composition with the ability to illicit a specific immune response against numerous cancer types. Further, when cultured with Peripheral blood mononuclear cells (PBMCs), monocytes, or dendritic cells, the resulting allogeneic autophagosome enriched compositions may induce or upregulate expression of BDCA3, and/or CD80, and further may induce secretion of IL-8 or other cytokine by PBMC.